Alphaviruses are pathogens of humans and livestock with worldwide distribution. The classification of some species as select agents in conjunction with their impact on public health makes understanding their interaction with the host and development of interventions a high priority. A major impediment to understanding the host cell components with which the virus interacts has been the inability to genetically manipulate the host cell/organism. Drosophila melanogaster represents an excellent experimental system for elucidating the genetic, molecular, and biochemical mechanisms underlying numerous physiological and developmental processes. The proposed research aims to exploit the power of Drosophila genetics for the discovery of host factors required for alphavirus genome replication. The basic concept for the research is straightforward;an alphavirus replicon sequence encoding a reporter/selectable marker will be cloned into the Drosophila genome under the control of the UAS/GAL4 system. Flies containing the replicon sequence will be crossed with a GAL4 expressing line to direct primary transcription of the replicon RNA in F1 progeny. If the cellular environment in which the replicon is expressed is permissive for virus genome replication then replicon amplification and reporter gene expression will occur. This approach will allow (i) the identification of tissues throughout the fly capable of supporting virus replication;(ii) genetic identification of host factors required for viral genome replication. The research outlined in the proposal is not complex, involving relatively simple manipulations of the Drosophila and alphaviral genomes;however the potential payoff is enormous. This simple but powerful approach will lead to the identification of dozens if not hundreds of host factors required for viral genome replication, and significantly advance our understanding of the virus-host interaction. PUBLIC HEALTH RELEVANCE: Identifying the ways in which a virus interacts with it's host is crucial to understanding the underlying cause of disease and developing antiviral drugs. The proposed research aims to determine host factors required for alphavirus (mosquito-borne pathogens) infection using a model genetic system.